1,3-propanediol is a monomer use in the production of polyester fibers and with potential use in the manufacture of polyurethanes and cyclic compounds.
1,3-propanediol can be produced by different chemical routes from i) acrolein water and hydrogen ii) ethylene oxide carbon monoxide and water in the presence of phosphine and from glycerol and hydrogen in the presence of carbon monoxide. All these methods have in common to be expensive and to generate waste streams containing polluting substances.
1,3-propanediol can be produced as an acetate/butyrate/lactate/1,3-propanediol mixture by the fermentation of glycerol by different Clostridia. The general metabolism of glycerol into Clostridia is presented in FIG. 1.
In one way, glycerol is converted to 1,3-propanediol in a two step enzymatic reaction sequence. In a first step a glycerol dehydratase catalyze the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA) and water. In the second step 3-HPA is reduced to 1,3-propanediol by a NADH dependent 1,3-propanediol dehydrogenase. Most of the 1,3-propanediol producing clostridia use a B12 dependent glycerol dehydratase encoded by the dhaB1B2B3 structural genes while Clostridum butyricum uses a B12 independent enzyme encoded by the dhaB1 structural gene. For the B12 dependent glycerol dehydratases, orfX and orfZ encode the glycerol dehydratase reactivation factor while for the only known B12 independent enzyme, dhaB2 encodes an S-Adenosyl-Méthionine (SAM) dependent activation factor. Near the genes encoding the structural and activation factors a gene encoding a 1,3-propanediol dehydrogenase (dhaT) is also present. Production of 1,3-propanediol from glycerol consumes NADH.
In another way, when glycerol is not transformed into 1,3-propanediol, it is oxidized to dihydrohycetone-phosphate (DHAP) with the concomitant production of NADH by either a glycerol kinase and a glycerol-3-Phosphate dehydrogenase encoded respectively by glpk and glpA or by a glycerol dehydrogenase followed by a DHA kinase encoded respectively by dhaD and dhaK1K2. DHAP will then enter the glycolitic pathway with the production of pyruvate and acetyl-CoA as key intermediates. Pyruvate and acetyl-CoA can be reduced to respectively lactate and ethanol by a lactate dehydrogenase encoded by the ldh gene and a bi-functional aldehyde-alcohol dehydrogenases encoded by adhE. Acetyl-CoA can also be converted to butyryl-CoA, an intermediate product that can be:                i) converted to butyric acid by a phospho-transbutyrylase and a butyrate kinase encoded respectively by the ptb and buk genes or        ii) reduced to butanol by a bi-functional aldehyde-alcohol dehydrogenase encoded by adhE.        
In solventogenic clostridia, acetone is produced from aceto-acetyl-CoA (an intermediate in the production of butyryl-CoA) by a CoA-transferase and an acetoacetate decarboxylase encoded respectively by the ctfAB and adc genes. Hydrogen is produced by an iron only hydrogenase encoded by the hydA gene.
Both natural and recombinant clostridia produce 1,3-propanediol at a maximal yield of 0.55 g/g of glycerol due to the co-production of reduced compounds like butyric acid (butyrate), lactic acid (lactate), ethanol or butanol. To increase the yield of 1,3-propanediol production it is necessary to avoid the production of all the reduced co-products and associate the production of 1,3-propanediol to an oxidized co-product.
Clostridium acetobutylicum strains unable to produce butyrate have already been described in the article (Green et al., 1996). The butyrate formation was dramatically reduced because of the inactivation of the buk gene obtained by single crossing-over with a non-replicable plasmid. This mutant strain was tested for the production of 1,3-propanediol as shown in (Gonzalez-Pajuelo, 2005, Metabolic Engineering). This recombinant strain effectively produces 1,3-propanediol as the main fermentation product, but produces also butanol, which decreases the 1,3-propanediol yield.
The 1,3-propanediol fermentation of glycerol by Clostridia can run in batch, fedbatch or continuous cultures.
The problem to be solved by the present invention is the biological production of 1,3 propanediol from glycerol at high yield, with no concomitant production of reduced compounds such as butyrate, lactate, or alcohols. This production is performed by anaerobic fermentation with Clostridia. 